PCMT1 regulates the migration, invasion, and apoptosis of prostate cancer through modulating the PI3K/AKT/GSK-3β pathway

Abstract

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins. Studies have shown that PCMT1 is overexpressed in several tumors and affects patients' prognosis. However, there are few reports on the role of PCMT1 in prostate cancer (PCa). In the present research, with the assistance of The Cancer Genome Atlas Program (TCGA) database, we found that PCMT1 was overexpressed in PCa tissues. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry staining also showed that PCMT1 expression was significantly increased in PCa tissues and cell lines. In PCa clinical samples, PCMT1 expression was closely related to Gleason score, clinical stage, lymph node metastasis and bone metastasis. The experiments of overexpression and knockdown of PCMT1 in vitro or in vivo showed that PCMT1 can significantly promote the proliferation, migration and invasion of PCa cells, inhibit cell apoptosis, and promote the growth of PCa. We furthermore confirmed that PCMT1 regulated the migration, invasion and apoptosis of PCa cells by modulating the phosphatidylinositol 3-kinase/AKT kinase/glycogen-synthase kinase-3β (PI3K/AKT/GSK-3β) signaling pathway. Collectively, PCMT1 plays a cancer-facilitative role in PCa by promoting the proliferation, migration and invasion of PCa cells, and inhibiting apoptosis. Therefore, PCMT1 is considered to represent a novel target for treating PCa.

Keywords: PCMT1; PI3K/AKT/GSK-3β signaling pathway; prostate cancer.

Figure 1

Expression of PCMT1 in PCa tissues and cell lines. (A) The expression of PCMT1 was identified in 492 PCa and 152 normal prostate tissues from TCGA and GTEx data. (B) PCMT1 protein expression in BPH and PCa tissues (IHC, ×400). (C) PCMT1 mRNA expression in PCa cell lines and the prostate epithelial cell line. (D) PCMT1 protein expression in PCa cell lines and normal prostate epithelial cell line. (E) Quantitative analysis of PCMT1 protein expression. Data are expressed as mean ± SD of at least three experiments. ^*P < 0.05 and ^***P < 0.001.

Figure 2

PC3 and DU145 cells transfected with siRNA-PCMT1 down-regulated PCMT1 expression and Flag-PCMT1 up-regulated PCMT1 expression. (A, B) siRNA-PCMT1 transfection down-regulated PCMT1 mRNA expression of PC3 and DU145 cells, respectively. (C, D) siRNA-PCMT1 transfection down-regulated PCMT1 protein expression of PC3 and DU145 cells. (E, F) Flag-PCMT1 transfection up-regulated PCMT1 protein expression of PC3 and DU145 cells. Data are presented as mean ± SD of at least three experiments. ^**P < 0.01 and ^***P < 0.001.

Figure 3

Impact of PCMT1 inhibition on the proliferation and apoptosis of PCa cells. (A, B) Effect of PCMT1 inhibition and overexpression on PCa cell proliferation measured by a CCK-8 assay or EdU assay. (C) Effect of PCMT1 inhibition and overexpression on PCa cell proliferation measured by a colony formation experiment. (D) Effect of PCMT1 inhibition and overexpression on PCa cell apoptosis measured by a flow cytometry assay. Data are expressed as mean ± SD of at least three experiments. ^***P < 0.001.

Figure 4

Impact of PCMT1 inhibition on the migration and invasion of PCa cell lines. (A) Effect of PCMT1 inhibition on PCa cell migration measured by wound-healing assay (×40). (B) PCa cell migration ability was assessed by a transwell migration assay (×200). (C) PCa cell invasion ability was examined by a transwell invasion assay (×200). Data are expressed as mean ± SD of at least three experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

PCMT1 regulates the EMT and apoptosis of PCa cells by modulating the PI3K/AKT/GSK-β signaling pathway. (A) Protein expression of factors involved in EMT, and quantification of the protein levels. (B) Protein expression of factors involved in apoptosis, and quantification of the protein levels. (C) Protein expression of P-AKT, AKT, P-GSK-3β, and GSK-3β, and quantification of the protein levels. Data are expressed as mean ± SD of at least three experiments. ^**P < 0. 01, ^***P < 0. 001.

Figure 6

PI3K/AKT signaling pathway activator 740Y-P affects the protein expression of related factors in siRNA-PCMT1 PCa cells. (A) Protein expression of E-cadherin, N-cadherin, Snail, Bax, Bcl-2, cleaved caspase-3, P-AKT, AKT, P-GSK-3β, and GSK-3β; in PC3 and DU145 cell lines. (B, C) Quantitative analysis of the protein expression levels in PC3 and DU145 cell lines. Data are expressed as mean ± SD of at least three experiments. ^**P < 0. 01, ^***P < 0. 001. Abbreviation: n. s: not significant.

Figure 7

PI3K activator 740Y-P can reverse the effect of siRNA-PCMT1 on the migration, invasion, and apoptosis of PCa cell lines. (A) The migration ability of PCa cells was measured by a transwell migration assay (×200). (B) The invasion ability of PCa cells was measured by a transwell invasion assay (×200). (C) The apoptotic rate of PCa cells was measured by a flow cytomet assay. Data are expressed as mean ± SD of at least three experiments. ^***P < 0. 001.

Figure 8

PCMT1 can affect the proliferation of prostate cancer cells in vivo. (A) Comparison of tumor tissue size in different groups of nude mice. (B) Comparison of tumor weights in different groups of nude mice. (C) The tumor volume was calculated at each time point. (D) PCMT1 protein expression in nude mice tumor tissues (IHC, ×200). Data are expressed as mean ± SD of at least three experiments. ^***P < 0. 001.

Figure 9

Schematic representation of the mechanism through which PCMT1 regulated EMT and apoptosis in PCa cells via PI3K/AKT/GSK-3β pathway.